This invention relates to methods and apparatus for measuring a property such as mass, size or density of target particles, and more particularly to such measurements using a Suspended Microchannel Resonator (SMR) and measuring the change in such properties over time and in changing environments.
Precision measurements of nanometer- and micrometer-scale particles, including living cells, have wide application in pharmaceuticals/drug delivery, disease studies, paints and coatings, foods, and other major industries and fields of research. This need is growing due to the expanding use of particulate engineering across these industries, to emerging nano- and micro-particle manufacturing techniques, to the need to better understand and treat diseases, and to recent regulations governing quality control in the pharmaceutical industry.
A variety of particle sizing and counting techniques, such as light scattering, Coulter Counters and others are known in the art. These techniques are embodied in commercial instruments and are used in industrial, medical, and research applications. Although such techniques have proven utility, they have limitations, which limit their applicability. Relatively recently, particle detection and measurement based on the use of SMR's has been developed, and shows promise of going beyond some of the limitations of conventional techniques. The SMR uses a fluidic microchannel embedded in a resonant structure, typically in the form of a cantilever or torsional structure. Fluids, possibly containing target particles are flowed through the sensor, and the contribution of the flowed material to the total mass within the sensor causes the resonance frequency of the sensor to change in a measurable fashion. SMR's are typically microfabricated MEMS devices. The use of microfabricated resonant mass sensors to measure fluid density has been known in the literature for some time [P. Enoksson, G. Stemme, E. Stemme, “Silicon tube structures for a fluid-density sensor”, Sensors and Actuators A 54 (1996) 558-562]. However, the practical use of resonant mass sensors to measure properties of individual particles and other entities suspended in fluid is relatively recent, as earlier fluid density sensors were not designed to measure individual particles at the micron and submicron scale.
In a body of work by common inventors and owned by the assignee of the current application, miniaturization and improvement of several orders of magnitude in mass resolution has been demonstrated. Development in the microfabrication recipes, the fluidics design, and measurement techniques are described in a number of co-pending patent applications and scientific publications. In particular U.S. patent application Ser. Nos. 11/620,320, 12/087,495, and 12/305,733 are particularly relevant and are incorporated by reference in their entirety. Also of relevance is a publication by some of the current inventors, [T. P. Burg, M. Godin, S. M. Knudsen et al., “Weighing of biomolecules, single cells and single nanoparticles in fluid,” Nature 446 (7139), 1066-1069 (2007)] By using the microfabrication techniques described in the references, SMR sensors have been fabricated with mass resolution of less than 1 femtogram (10−15 g). This resolution is sufficient to detect and measure the mass of individual particles in the range of ˜100 nanometers up to many microns in size, including mammalian cells.
Improvements in SMR based measurement techniques have been disclosed, particularly in the parent application of this application, allow for a particle to remain in the measurement portion of the SMR for extended periods of time. Although the disclosed techniques have the advantage of improving signal to noise, they also provide for the ability to measure particle properties which may change over time. Of particular interest is the possibility of measuring cell growth. High precision measurements of the mass of living cells have not been possible previously. Three methods are commonly used to measure cell size and none can measure mass. In the first, cross-sectional areas derived from focused microscope images are used to estimate cell volume, through either integration of a series or assumption of a simple shape (e.g. a sphere). In the second, the forward scatter (FSC) of light by cells is measured. In the third, a particle analyzer, the Coulter Counter, uses volume displacement of an electrolyte to measure cell volume. The first two approaches suffer from a lack of precision and in addition, FSC measurements are not based on an absolute scale. Nor is it clear how linear the relationship between FSC and cell size actually is, or how dependent FSC is on additional physical properties. The third approach (volume displacement) is the most effective but requires the dangerous assumption that mass density is constant. Eukaryotic cell volume and hence mass density can change quickly by altering ion balance. Even in cells with rigid cell walls (e.g. yeast), mass density depends on the proportion of volume occupied by large fluid-filled vacuoles, a proportion sensitive to environmental and genetic alterations.
Given the mass resolution of current SMR's cell mass measurements potentially may be accomplished at very high resolution, for a single mammalian cell approximately 0.01%, which is orders of magnitude better than what could be achieved with existing optical methods. With such resolution it is possible to measure cell mass change over time and potentially even more importantly in response to changes in the chemical or environmental properties of the cell's liquid environment. Such measurements would have applicability in drug resistance/susceptibility studies, and general environmental toxicity studies. For example, clinical oncology and cancer biology are challenged by the lack of assay platforms for measuring changes in cancer-cells' growth kinetics in response to chemical therapeutic intervention. Therefore it is the object of this invention to disclose methods and apparatus for extended time measurements of particles in an SMR along with methods and apparatus to change the chemical or environmental properties of the fluids in extended time measurements.